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ABSTRACT Achieving targeted perturbations of neural activity is essential for dissecting the causal architecture of brain circuits. A crucial challenge in targeted manipulation experiments is the identification ofhigh efficacyperturbation sites whose stimulation exerts desired effects, currently done with costly trial-and-error procedures. Can one predict stimulation effects solely based on observations of the circuit activity, in the absence of perturbation? We answer this question in dissociated neuronal cultures on High-Density Microelectrode Arrays (HD-MEAs), which, compared toin vivopreparations, offer a controllablein vitroplatform that enables precise stimulation and full access to network dynamics. We first reconstruct theperturbome- the full map of network responses to focal electrical stimulation - by sequentially activating individual single sites and quantifying their network-wide effects. The measured perturbome patterns cluster into functional modules, with limited spread across clusters. We then demonstrate that the perturbome can be predicted from spontaneous activity alone. Using short baseline recordings in the absence of perturbations, we estimate Effective Connectivity (EC) and show that it predicts the spatial organization of the perturbome, including spatial clusters and local connectivity. Our results demonstrate that spontaneous dynamics encode the latent causal structure of neural circuits and that EC metrics can serve as effective, model-free proxies for stimulation outcomes. This framework enables data-driven targeting and causal inferencein vitro, with potential applications to more complex preparations such as human iPSC-derived neurons and brain organoids, with implications for both basic research and therapeutic strategies targeting neurological disorders. Significance StatementNeuronal cultures are increasingly used as controllable platforms to study neuronal network dynamics, neuromodulation, and brain-inspired therapies. To fully exploit their potential, we need robust methods to probe and interpret causal interactions. Here, we develop a framework to reconstruct the perturbome—the network-wide map of responses to localized electrical stimulation—and show that it can be predicted from spontaneous activity alone. Using simple, model-free metrics of Effective Connectivity, we reveal that ongoing activity encodes causal structure and provides reliable proxies for stimulation outcomes. This validates EC as a practical measure of causal influence in vitro. Our methodology refines the use of neuronal cultures for brain-on-a-chip approaches, and paves the way for data-driven neuromodulation strategies in human stem cell–derived neurons and brain organoids.
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This content will become publicly available on May 30, 2026
Towards model-based design of causal manipulations of brain circuits with high spatiotemporal precision
Abstract Recent advancements in neurotechnology enable precise spatiotemporal patterns of micros- timulations with single-cell resolution. The choice of perturbation sites must satisfy two key criteria: efficacy in evoking significant responses and selectivity for the desired target effects. This choice is currently based on laborious trial-and-error procedures, unfeasible for sequences of multi-site stimulations. Efficient methods to design complex perturbation patterns are ur- gently needed. Can we design a spatiotemporal pattern of stimulation to steer neural activity and behavior towards a desired target? We outline a method for achieving this goal in two steps. First, we identify the most effective perturbation sites, or hubs, only based on short observations of spontaneous neural activity. Second, we provide an efficient method to design multi-site stimulation patterns by combining approaches from nonlinear dynamical systems, control theory and data-driven methods. We demonstrate the feasibility of our approach using multi-site stimulation patterns in recurrent network models.
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- Award ID(s):
- 2238247
- PAR ID:
- 10631307
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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